Withania somnifera L. is a medicinal herb related to Solanaceae family, known as ashwagandha. The aqueous extract of W. somnifera was utilized for the green synthesis of zinc nanoparticles to investigated their antioxidant and anticancer potentials. The synthesized zinc nanoparticle solution of W. somnifera extract was characterized by phytochemical analysis, Transmission Electron Microscope (TEM), and zeta potential. The results revealed reduced values of the phytochemical constitutes, large surface area, and high stability of the nanoparticles. The antioxidant activity of the extracted W. somnifera and its zinc nanoparticles was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH•) assay, in which the original extract has the more antioxidant capacity with IC50= 0.701 mg/mL, along with potent results for both samples relative to ascorbic acid. The extracted W. somnifera, its zinc nanoparticles, and zinc sulfate solution were in vitro assessed as anticancer agents on well-known six human tumor, and a normal cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The results demonstrated that the synthesized zinc nanoparticles of the W. somnifera extract shown the most potent cytotoxicity with IC50= 19.17 µg/mL on HeLa cell line. The synthesized nano-zinc solution of the W. somnifera aqueous extract performed to be proficient as a potent anticancer characteristic than the plant extract with developed biological potency.